Neurotransmitter detection method based on a continuous reaction platform of integrated derivatization and solid phase extraction

Abstract

The present invention discloses a neurotransmitter detection method based on an integrated derivatization and solid phase extraction continuous reaction platform; the neurotransmitters include at least: Class A neurotransmitters have no primary amine groups, hydroxyl groups, and carboxyl groups, and have secondary amine groups or quaternary ammonium groups; Class B neurotransmitters contain at least one of primary amine groups, hydroxyl groups, and carboxyl groups; before detection, an aqueous phase derivatization system containing benzoyl chloride is used to derivatize the neurotransmitters in the sample, and an aqueous formic acid solution is added to the obtained supernatant to mix and centrifuge, and the obtained supernatant is subjected to LC-MS analysis after solid phase extraction. The present invention forms an integrated derivatization and solid phase extraction continuous reaction platform through process design, and realizes high-sensitivity simultaneous detection of at least two types of neurotransmitters. Melatonin, serotonin, histamine, and dopamine are detected simultaneously with high sensitivity, and the detection sensitivity is 1pg / mL for melatonin, 1ng / mL for serotonin, 0.03ng / mL for histamine, and 10pg / mL for dopamine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomarker detection, and relates to a neurotransmitter detection method based on an integrated derivatization and solid phase extraction continuous reaction platform, and in particular to a method for simultaneously detecting melatonin, serotonin, histamine and dopamine with high sensitivity based on an integrated derivatization and solid phase extraction continuous reaction platform. Background Art

[0002] Melatonin, serotonin, histamine, and dopamine are very important compounds in the nervous system. These compounds play a key role in the nervous system, each regulating different physiological and psychological processes. Melatonin: Secreted by the pineal gland, it mainly regulates the sleep-wake cycle and biological rhythms. Abnormal melatonin levels are associated with insomnia, depression, and seasonal affective disorder. Serotonin: It is distributed in the brain and intestines and is involved in regulating emotions, appetite, sleep, and memory. Serotonin deficiency is associated with depression, anxiety, and other mood disorders. Histamine: As a neurotransmitter in the central nervous system, it regulates wakefulness, appetite, and immune responses. Histamine is also involved in allergic and inflammatory reactions. Dopamine: Regulates movement, reward mechanisms, motivation, and pleasure. Dopamine deficiency is associated with Parkinson's disease and depression, while excess is associated with schizophrenia and drug dependence.

[0003] Simultaneous detection of melatonin, serotonin, histamine, and dopamine can provide comprehensive neurochemical status information, which is helpful for: 1) Comprehensive diagnosis: Through comprehensive analysis of multiple neurotransmitter levels, it helps to diagnose neurological diseases such as depression, anxiety, Parkinson's disease, etc. 2) Precision treatment: Monitor changes in multiple neurotransmitters, optimize drug treatment plans, and improve treatment effects. 3) Biomarker discovery: Multi-parameter detection can discover new biomarkers for early diagnosis and prognosis assessment.

[0004] There are many technical challenges in detecting these neurotransmitters at the same time: 1) Melatonin and serotonin, histamine, and dopamine have quite different core functional groups. Melatonin has no active primary amino or hydroxyl groups. The amino and hydroxyl groups in the structure of melatonin have been replaced, resulting in only secondary amine groups. Secondary amine groups are chemically stable and cannot undergo rapid derivatization reactions at room temperature unless there are very intense conditions. Serotonin, histamine, and dopamine all have active primary amine groups, hydroxyl groups, and carboxyl groups. These chemical groups can easily undergo rapid derivatization reactions at room temperature. 2) The content of compounds such as melatonin, serotonin, histamine, and dopamine in the body is very low, and the detection sensitivity requirements are very high. Taking melatonin as an example, the lowest concentration of melatonin in healthy people during the day reaches 2pg / mL. Therefore, the detection technology must be able to measure concentrations below 2pg / mL to meet the requirements of clinical detection. Otherwise, a large number of people will not be able to detect melatonin in their plasma. Healthy people cannot detect it, and special patients will have even lower melatonin levels, which makes it even more difficult to judge, and therefore cannot meet the detection requirements. Serotonin, histamine, and dopamine also have similar problems. Because of the special biological characteristics of neurotransmitters, they require very high sensitivity in detection. It is difficult for previous methods to meet the detection requirements because their actual applications have not been carried out; 3) Melatonin, serotonin, histamine, and dopamine are the most critical indicators. Clinically, they are required to be detected together. It is relatively easy to detect these compounds alone. Each compound can select the most suitable method for them and carry out a large number of targeted optimizations with the goal of improving sensitivity. Unfortunately, if these compounds are detected alone, the clinical report time "TAT" will be greatly increased. "TAT" (Turnaround Time) means the time required from receiving the sample to issuing the report. The TAT requirements for these compounds are very high because these compounds change very quickly in the body and must be detected as soon as possible. Multiple tests cannot meet the clinical TAT requirements at all.

[0005] Except for melatonin, the LogP values ​​of other compounds are very low or even negative, which means they cannot be extracted by conventional LLE or SPE.

[0006] name LogP (lipophilicity) Melatonin 1.48 serotonin -0.36 histamine -0.57 Dopamine -0.61

[0007] The structural formulas of melatonin, serotonin, histamine, and dopamine are as follows:

[0008]

[0009] There have been reports on the use of benzoyl chloride for derivatization reactions, but there have been no public reports on the simultaneous determination of neurotransmitters such as melatonin, serotonin, histamine, and dopamine using a continuous reaction platform that integrates derivatization and solid phase extraction.

[0010] After searching the existing patent documents, it was found that CN201510076850.4 discloses a pretreatment method for biogenic amine neurotransmitters and / or their metabolites, including derivatizing a sample containing biogenic amine neurotransmitters and / or their metabolites, and performing online solid phase extraction of the derivatized products; wherein the structure of the derivatization reagent contains a sulfonyl chloride group or an acyl chloride group connected to an aromatic ring; the biogenic amine neurotransmitters or their metabolites are one or more of dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine and kynurenine. The derivatization reactions used in this patent all require 37°C and cannot be carried out at room temperature. According to the description in the patent, the reaction time is specifically 37°C and 40 minutes (page 11, the rest of the embodiments are the same as embodiment 1). Non-room temperature reactions, especially such a long reaction time, are far from meeting clinical requirements, will greatly extend the reporting time, and cannot be used for the detection of temporary samples. Moreover, in this patent, the sensitivity of detection still does not meet the requirements of clinical detection at non-room temperature and such a long reaction time. For example, the level of dopamine in healthy people is below 30pg / mL, and many people are around 10pg / mL. The linear range of 1-1000ng / mL in this patent means that the linearity can only cover the lowest 1ng / mL, that is, 1000pg / mL, which is 100 times different from the clinical detection requirement of 10pg / mL. Furthermore, the content of melatonin in healthy people is very low, and many people are below 5pg / mL. Therefore, a concentration of 1-2pg / mL needs to be detected to meet clinical requirements. The melatonin in this patent appears in Examples 3 and 4, and the linear range is 1-1000ng / mL, which means that the linearity can only cover the lowest 1ng / mL, that is, 1000pg / mL, which is 1000 times different from the clinical detection requirement.

[0011] CN202010327051.0 discloses a method for detecting hormones and neurotransmitters in serum; among them, the quantitative limit of dopamine is 0.23ng / mL, i.e. 230pg / mL, which is far from the 10pg / mL required for clinical testing. Similarly, the quantitative limit of melatonin is 0.06ng / mL, i.e. 60pg / mL, which is still far from the 1pg / mL required for clinical testing. Summary of the invention

[0012] The purpose of the present invention is to provide a neurotransmitter detection method based on an integrated derivatization and solid phase extraction continuous reaction platform, and in particular to a method for high-sensitivity simultaneous detection of melatonin, serotonin, histamine and dopamine based on an integrated derivatization and solid phase extraction continuous reaction platform. The present invention forms an integrated derivatization and solid phase extraction continuous reaction platform through process design, realizes high-sensitivity simultaneous detection of melatonin, serotonin, histamine and dopamine, and realizes detection sensitivities of 1pg / mL for melatonin, 1ng / mL for serotonin, 0.03ng / mL for histamine and 10pg / mL for dopamine.

[0013] The objective of the present invention is achieved through the following technical solutions:

[0014] The present invention provides a neurotransmitter detection method based on an integrated derivatization and solid phase extraction continuous reaction platform, wherein the neurotransmitters include at least two categories: Class A neurotransmitters have no primary amine groups, hydroxyl groups, and carboxyl groups; Class B neurotransmitters contain at least one of primary amine groups, hydroxyl groups, and carboxyl groups;

[0015] Before the test, the neurotransmitters in the plasma or serum samples were derivatized using an aqueous derivatization system containing benzoyl chloride, and an aqueous formic acid solution was added to the obtained supernatant, mixed and centrifuged. The obtained supernatant was subjected to solid phase extraction and then analyzed by LC-MS.

[0016] As an embodiment of the present invention, the class A neurotransmitter includes melatonin; the class B neurotransmitter includes at least one of serotonin, histamine, dopamine, γ-aminobutyric acid, glutamine, 5-hydroxyindole-3-acetic acid, levodopa, kynurenine, tryptamine, glutamate, and tryptophan.

[0017] As an embodiment of the present invention, the neurotransmitters include melatonin, serotonin, histamine, and dopamine.

[0018] As an embodiment of the present invention, the derivatization treatment comprises the following steps:

[0019] A1. Take a plasma or serum sample, add an internal standard solution and a zinc sulfate aqueous solution, mix well, centrifuge and take the supernatant;

[0020] A2, add sodium carbonate aqueous solution and mix well;

[0021] A3. Continue to add acetonitrile solution of benzoyl chloride, mix well and centrifuge.

[0022] As one embodiment of the present invention, the concentration of the aqueous zinc sulfate solution is 100-300 mM;

[0023] and / or, the concentration of the sodium carbonate aqueous solution is 1000mM-2000mM;

[0024] And / or, the concentration of the acetonitrile solution of benzoyl chloride is 10%-20%.

[0025] As an embodiment of the present invention, the volume ratio of the sample to the aqueous zinc sulfate solution is 100:100-400;

[0026] and / or, the volume ratio of the sample to the sodium carbonate aqueous solution is 100:50-100;

[0027] And / or, the volume ratio of the sample to the acetonitrile solution of benzoyl chloride is 100:20-100.

[0028] As an embodiment of the present invention, the mixing and centrifugation is vortex mixing (rotation speed 1000-1500 rpm) and high-speed centrifugation (14,000 rpm, 4° C.).

[0029] As an embodiment of the present invention, the internal standard in the internal standard solution is an isotope internal standard of a neurotransmitter.

[0030] The volume ratio of the sample to the internal standard solution is 100:10-50. As one embodiment of the present invention, the internal standard solution contains isotope internal standards of melatonin, serotonin, histamine, and dopamine, with concentrations of 0.05-0.5 ng / mL, 50

[0031] In some specific embodiments, the internal standard solution contains isotopic internal standards of melatonin, serotonin, histamine, and dopamine, such as melatonin-D4, 5-hydroxytryptamine-D4, histamine-D4, and dopamine-D4, with concentrations of 0.05 ng / mL, 50 ng / mL, 1.5 ng / mL, and 0.5 ng / mL, respectively.

[0032] As one embodiment of the present invention, the concentration of the formic acid aqueous solution is 1M-3M.

[0033] As an embodiment of the present invention, the volume ratio of the sample to the formic acid aqueous solution is 100:30-100.

[0034] As an embodiment of the present invention, the solid phase extraction is to load the obtained supernatant onto a C18 matrix SPE plate, and after elution, acetonitrile is added for elution.

[0035] As one embodiment of the present invention, the loading volume of the supernatant is 180-380 μl; and / or, 100-200 μl of 90-100% acetonitrile is added for elution.

[0036] As one embodiment of the present invention, the content of neurotransmitters in the sample is calculated based on the standard curve method in combination with LC-MS analysis.

[0037] In some implementation examples, during LC-MS analysis, the liquid phase conditions are as follows:

[0038] Chromatographic conditions:

[0039] Mobile phase A: methanol

[0040] Mobile phase B: 0.1% formic acid in water

[0041] Gradient conditions:

[0042] Time / minute Phase A / % Phase B / % Gradient Curve 0 98 5 / 9.9 5 95 6 10.0 0 100 6 11.0 0 100 6 12.0 98 5 1 .

[0043] In some implementation examples, during LC-MS analysis, the mass spectrometry conditions are as follows:

[0044]

[0045] It should be noted that in the embodiments of the present invention, only melatonin, serotonin, histamine, and dopamine were selected to display the continuous reaction platform of integrated derivatization and solid phase extraction for high-sensitivity and simultaneous detection of this type of compounds. The same method can be used for the detection of other similar compounds, including but not limited to γ-aminobutyric acid, glutamine, 5-hydroxyindole-3-acetic acid, L-dopa, kynurenine, tryptamine, glutamate, tryptophan, etc.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1) Through process design, an integrated derivatization and solid phase extraction continuous reaction platform was formed, which achieved high-sensitivity simultaneous detection of melatonin, serotonin, histamine, and dopamine, and achieved detection sensitivities of 1 pg / mL for melatonin, 1 ng / mL for serotonin, 0.03 ng / mL for histamine, and 10 pg / mL for dopamine.

[0048] 2) Select a specific derivatization reagent that can react quickly at room temperature, and first use the derivatization method to improve the detection sensitivity of serotonin, histamine, and dopamine.

[0049] 3) The traditional derivatization system was changed to connect non-polar groups (benzoyl groups) to serotonin, histamine, and dopamine. The reaction environment of the traditional derivatization system is an organic phase-based reaction system, which causes serious loss of melatonin and other compounds in the subsequent steps. Changing the derivatization system from an organic phase system to an aqueous phase system greatly improves the detection sensitivity of melatonin, serotonin, histamine, and dopamine.

[0050] 4) Changed the high pH value and high ionic strength at the end of the traditional derivatization system. The traditional derivatization system ends with a high pH environment and high ionic strength. The high pH environment and high ionic strength will affect the detection sensitivity of melatonin, serotonin, histamine, and dopamine in the subsequent steps. By adding reagents to adjust to the optimal pH value and reducing the ionic strength, the sensitivity is greatly improved in combination with SPE.

[0051] 5) By adding a large number of non-polar groups to the structure and matching the appropriate derivatization system and the system before SPE loading, the disadvantage of serotonin, histamine, and dopamine not being retained on the C18 matrix SPE was changed, so that these compounds can be well retained on the C18 matrix SPE. The combination of derivatization and SPE, two technologies that improve sensitivity and purification, enables melatonin, serotonin, histamine, and dopamine to achieve ultra-high sensitivity.

[0052] 6) The design of the continuous reaction platform for integrated derivatization and solid phase extraction enables derivatization and SPE to be completed in one pot. Only various reagents need to be added. No drying, liquid-liquid extraction, or desalting is required during the process, thus achieving high-sensitivity one-pot determination of melatonin, serotonin, histamine, and dopamine for one sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0054] Figure 1 Schematic diagram of the continuous reaction platform of one-pot derivatization and solid phase extraction;

[0055] Figure 2 This is a typical chromatogram of melatonin, serotonin, histamine and dopamine; from top to bottom, they are dopamine, histamine, serotonin and melatonin;

[0056] Figure 3 This is the chromatogram of melatonin at the lowest point of the standard curve (retention time 5.37 minutes);

[0057] Figure 4 This is the chromatogram of serotonin at the lowest point of the standard curve (retention time 7.87 minutes);

[0058] Figure 5 The chromatogram of histamine at the lowest point of the standard curve (retention time 3.80 minutes);

[0059] Figure 6 This is the chromatogram of dopamine at the lowest point of the standard curve (retention time 8.70 minutes);

[0060] Figure 7 is the standard curve of melatonin;

[0061] Figure 8 is the serotonin standard curve;

[0062] Fig. 9 is the histamine standard curve;

[0063] Fig.10 is the dopamine standard curve;

[0064] Fig.11 This is the dopamine chromatogram after adding acetonitrile in step 2 of the first part of the main working mechanism, and no obvious chromatographic peak is observed;

[0065] Fig.12 is the change in the peak area of ​​dopamine under different concentrations of zinc sulfate aqueous solution (the abscissa corresponds to the number of different zinc sulfate concentrations in Table 1, and the ordinate is the peak area of ​​dopamine);

[0066] Fig.13 is the change in the peak area of ​​dopamine under different sodium carbonate solution concentrations (the abscissa corresponds to the number of different sodium carbonate solution concentrations in Table 2, and the ordinate is the peak area of ​​dopamine);

[0067] Fig.14 is the change in the peak area of ​​dopamine at different benzoyl chloride concentrations (the abscissa corresponds to the number of different benzoyl chloride concentrations in Table 3, and the ordinate is the peak area of ​​dopamine);

[0068] Fig.15 is the change in the peak area of ​​dopamine under different addition amounts of benzoyl chloride (the abscissa corresponds to the addition amounts of different benzoyl chlorides in Table 4, and the ordinate is the peak area of ​​dopamine);

[0069] Fig.16 It is the change of the peak area of ​​dopamine when adding different formic acid concentrations (the abscissa corresponds to the different formic acid concentrations in Table 5, and the ordinate is the peak area of ​​dopamine). DETAILED DESCRIPTION

[0070] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0071] In the examples, if the specific conditions are not specified, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0072] The purpose of the present invention is to provide a neurotransmitter detection method based on an integrated derivatization and solid phase extraction continuous reaction platform, and in particular to a method for simultaneously detecting melatonin, serotonin, histamine and dopamine with high sensitivity based on an integrated derivatization and solid phase extraction continuous reaction platform. Figure 1 The method is exemplified as follows, including:

[0073] Step 1. Accurately pipette 100 μL of plasma (or serum or standard curve) sample and add 10 μL of internal standard solution (isotope internal standards of melatonin, serotonin, histamine, and dopamine, with concentrations of 0.05 ng / mL, 50 ng / mL, 1.5 ng / mL, and 0.5 ng / mL, respectively).

[0074] Step 2: Add 100-400 μl of zinc sulfate aqueous solution with a concentration of 100-300 mM, vortex to mix, centrifuge at high speed, and remove the supernatant.

[0075] Step 3: Add 50-100 μl of 1000 mM-2000 mM sodium carbonate aqueous solution to the supernatant in step 2 and vortex to mix.

[0076] Step 4: Add 20-100 μl of acetonitrile solution of benzoyl chloride (concentration of 10%-20%), vortex mix, and centrifuge at high speed.

[0077] Step 5: Add 100 μl of 1M-3M formic acid aqueous solution to the supernatant in step 4, vortex to mix, and centrifuge at high speed.

[0078] Step 6: Load the supernatant from step 5 onto a C18-based SPE plate, and after washing, add 100-200 μl of 90-100% acetonitrile for elution.

[0079] Step 7: Inject into LC-MS for analysis.

[0080] The liquid phase conditions are as follows:

[0081] Chromatographic conditions:

[0082] Mobile phase A: methanol

[0083] Mobile phase B: 0.1% formic acid in water

[0084] Gradient conditions:

[0085] Time / minute Phase A / % Phase B / % Gradient Curve 0 98 5 / 9.9 5 95 6 10.0 0 100 6 11.0 0 100 6 12.0 98 5 1 .

[0086] The mass spectrometry conditions were as follows:

[0087]

[0088]

[0089] The main working mechanisms involved in this method are described as follows:

[0090] Part 1: Select appropriate derivatization reagents. First, improve the detection sensitivity of serotonin, histamine, and dopamine through derivatization. Change the traditional derivatization system to connect non-polar groups to serotonin, histamine, and dopamine. The reaction environment of the traditional derivatization system is an organic phase-based reaction system. The organic phase-based system causes serious loss of melatonin and other compounds in the subsequent steps. Changing the derivatization system from an organic phase system to an aqueous phase system greatly improves the detection sensitivity of melatonin, serotonin, histamine, and dopamine.

[0091] The first part involves steps 2 to 4 above, and the detailed descriptions and data are as follows:

[0092] Step 2: Add 100-400 μl of zinc sulfate aqueous solution with a concentration of 100-300 mM, vortex to mix, centrifuge at high speed, and remove the supernatant.

[0093] Step 3: Add 50-100 μl of 1000 mM-2000 mM sodium carbonate aqueous solution to the supernatant in step 2 and vortex to mix.

[0094] Step 4: Add 20-100 μl of acetonitrile solution of benzoyl chloride (10%-20% by volume), vortex to mix, and centrifuge at high speed.

[0095] 1) Function: Replace the traditional derivatization system and use the aqueous phase system to replace the traditional organic phase system;

[0096] 2) Comparison and difference with traditional methods: Traditional derivatization systems use organic phase as the main reaction system. Although there is a small amount of water, the organic phase is the main body. In the present invention, the water phase is used as the main system with only a small amount of organic phase.

[0097] 3) Value: The traditional organic phase system will greatly reduce the sensitivity of the compound when integrated with the subsequent SPE. However, after changing to a derivatization system based on the aqueous phase, the sensitivity will be greatly improved.

[0098] 4) Key technology: In step 2, an aqueous solution of zinc sulfate is added at a concentration of 100-300 mM; in step 3, an aqueous solution of sodium carbonate is added at a concentration of 1000 mM-2000 mM in an amount of 50-100 microliters; in step 4, a concentration of 10-20% benzoyl chloride is added in an amount of 20-100 microliters;

[0099] A. First, the traditional organic phase system will cause a large amount of target compound loss. According to the above steps, take an equal amount of sample, except that acetonitrile is added instead of zinc sulfate in step 2. Specifically, step 1, accurately aspirate 100 microliters of the standard sample, add 10 microliters of internal standard solution (melatonin-D4, 5-hydroxytryptamine-D4, histamine-D4, dopamine-D4, concentrations are 0.05ng / mL, 50ng / mL, 1.5ng / mL, 0.5ng / mL respectively). Step 2, add 200 microliters of acetonitrile, vortex mix, high-speed centrifuge, and remove the supernatant. Step 3, add 100 microliters of 2000mM sodium carbonate aqueous solution to the supernatant of step 2, vortex mix. Step 4, add 100 microliters of acetonitrile solution of benzoyl chloride (concentration is 10%), vortex mix, and high-speed centrifuge. Step 5. Add 100 μl of 2M formic acid solution to the supernatant of step 4, vortex to mix, and centrifuge at high speed. Step 6. Load the supernatant of step 5 onto a C18-based SPE plate, rinse, and then add 200 μl of 100% acetonitrile to elute. Step 7. Inject into LC-MS for analysis. In this case, dopamine is almost undetectable. The following is the chromatogram of dopamine when an organic system (acetonitrile) is added. Fig.11 It can be seen that there is no obvious chromatographic peak for dopamine.

[0100] B. Follow the above steps, except that 200 μl of zinc sulfate aqueous solution is added in step 2; take an equal amount of sample, only change the concentration of zinc sulfate aqueous solution, and keep other conditions unchanged, and take the peak area of ​​dopamine (such as Fig.12) as an example (because dopamine is the most difficult compound to detect among neurotransmitters, as long as the detection performance of dopamine can meet the requirements, other compounds can meet the requirements, so the performance of dopamine is focused on during the investigation. Further, if the optimal conditions for dopamine are not the optimal conditions for other compounds, the optimal conditions for dopamine will also be used as the final use conditions for the joint detection of a group of compounds, because although for other compounds, it may not be its optimal condition, it can also meet the requirements of the determination. The results of all compounds under the optimal conditions are shown in Example 1.) The results are shown in Table 1 below, and the results show that the concentration of zinc sulfate needs to be 100-300mM to achieve optimal sensitivity.

[0101] Table 1

[0102] Number (shown on the horizontal axis in the figure) Zinc sulfate concentration (mM) Area 1 50 421410.0 2 100 1275546.7 3 200 2046380.4 4 250 1191586.1 5 300 1261932.6

[0103] C. According to the above steps (same as B), take the same amount of sample, only change the concentration and volume of sodium carbonate aqueous solution, and keep other conditions unchanged. Take the peak area of ​​dopamine ( Fig.13 ) as an example to show the results in Table 2 below. The results show that the concentration of sodium carbonate aqueous solution is 1000mM-2000mM, which can achieve the best sensitivity. The amount of sodium carbonate aqueous solution added should be 50-100 microliters:

[0104] Table 2

[0105]

[0106]

[0107] D. According to the above steps, take 8 equal amounts of samples, only change the concentration of benzoic acid chloride, and keep other conditions unchanged. Take the peak area of ​​dopamine ( Fig.14 ) as an example, the results are shown in Table 3 below, and the results prove that 10%-20% benzoyl chloride can efficiently derive serotonin, histamine, and dopamine in an aqueous system.

[0108] Table 3

[0109] Number (shown on the horizontal axis in the figure) Benzoyl chloride concentration Area 1 1％ 1154.7 2 2％ 1411.3 3 3％ 2950.7 4 4％ 3592.3 5 5％ 9108.0 6 10％ 54344.4 7 20％ 48752.6 8 40％ 20142.8

[0110] E. According to the above steps (same as B), take 4 equal amounts of samples, only change the amount of benzoyl chloride added (benzoyl chloride concentration is 10%), and keep other conditions unchanged. Take the peak area of ​​dopamine ( Fig.15 ) as an example, the results are shown in Table 4 below, and the results prove that 20-100 μL of benzoyl chloride can efficiently derive serotonin, histamine, and dopamine in an aqueous system.

[0111] Table 4

[0112] Number (shown on the horizontal axis in the figure) Amount of benzoyl chloride added (μl) Area 1 20 854405.6 2 50 917242.0 3 80 972834.7 4 100 831202.9

[0113] Part 2: Changing the high pH value and high ionic strength at the end of the traditional derivatization system. The traditional derivatization system ends with a high pH environment and high ionic strength. The high pH environment and high ionic strength will affect the detection sensitivity of melatonin, serotonin, histamine, and dopamine in the subsequent steps. By adding reagents to adjust to the optimal pH value and reducing the ionic strength, the sensitivity is greatly improved in combination with SPE.

[0114] The second part involves step five above, and the detailed description and data are as follows:

[0115] Step 5: Add 100 μl of 1M-3M formic acid aqueous solution to the supernatant in step 4, vortex to mix, and centrifuge at high speed.

[0116] 1) Function: Replace the high pH and high ionic strength at the end of the traditional derivatization system, and use the aqueous phase system to replace the traditional organic phase system;

[0117] 2) Comparison and difference with traditional methods: no liquid-liquid extraction, no desalting, no additional steps, and the most suitable reagent, 1M-3M formic acid aqueous solution, is added to reduce the pH value and ionic strength, simplifying the operation process to the greatest extent and realizing a one-pot method;

[0118] 3) Value: High pH environment and high ionic strength will affect the detection sensitivity of melatonin, serotonin, histamine, and dopamine in the subsequent steps. Adding 1M-3M formic acid aqueous solution changes the state of the system;

[0119] 4) Key technology: The reagent added is formic acid, the concentration of formic acid is 1M-3M (preferably 2M), and the amount of formic acid added is 100 μL;

[0120] E. According to the above steps, take the same amount of sample, only change the concentration of formic acid solution, and keep other conditions unchanged. Take the peak area of ​​dopamine ( Fig.16 ) as an example, the results are shown in Table 5 below, and the results show that the concentration of formic acid needs to be 1M-3M to achieve the best sensitivity.

[0121] Table 5

[0122] serial number Formic acid concentration (M) Area 1 0.2 30240.9 2 0.5 63969.5 3 1 86285.4 4 2 168223.4 5 3 59691.1

[0123] Example 1

[0124] A dopamine series standard curve was prepared with concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 ng / mL, respectively. A histamine series standard curve was prepared with concentrations of 0.03, 0.06, 0.15, 0.3, 0.6, 1.5, 3, 6, and 15 ng / mL, respectively. A serotonin series standard curve was prepared with concentrations of 1, 2, 5, 10, 20, 50, 100, 200, and 500 ng / mL, respectively. A melatonin series standard curve was prepared with concentrations of 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 ng / mL, respectively.

[0125] Follow the steps below to determine the linearity of melatonin, serotonin, histamine, and dopamine.

[0126] Step 1. Accurately aspirate 100 μL of plasma (or serum) sample and add 10 μL of internal standard solution (isotope internal standards of melatonin, serotonin, histamine, and dopamine: melatonin-D4, 5-hydroxytryptamine-D4, histamine-D4, and dopamine-D4, with concentrations of 0.05 ng / mL, 50 ng / mL, 1.5 ng / mL, and 0.5 ng / mL, respectively).

[0127] Step 2: Add 200 μL of zinc sulfate aqueous solution with a concentration of 200 mM, vortex to mix, centrifuge at high speed, and remove the supernatant.

[0128] Step 3: Add 100 μl of 2000 mM sodium carbonate aqueous solution to the supernatant in step 2 and vortex to mix.

[0129] Step 4: Add 100 μl of acetonitrile solution of benzoyl chloride (concentration is 10%), vortex mix, and centrifuge at high speed.

[0130] Step 5: Add 100 μl of 2 M formic acid aqueous solution to the supernatant in step 4, vortex to mix, and centrifuge at high speed.

[0131] Step 6: Load the supernatant from step 5 onto a C18-based SPE plate, wash, and then add 200 μL of 100% acetonitrile for elution.

[0132] Step 7: Inject into LC-MS for analysis.

[0133] The liquid phase conditions are as follows:

[0134] Chromatographic conditions:

[0135] Mobile phase A: methanol

[0136] Mobile phase B: 0.1% formic acid in water

[0137] Gradient conditions:

[0138] Table 6

[0139] Time / minute Phase A / % Phase B / % Gradient Curve 0 98 5 / 9.9 5 95 6 10.0 0 100 6 11.0 0 100 6 12.0 98 5 1

[0140] The mass spectrometry conditions were as follows:

[0141] Table 7

[0142]

[0143] Typical chromatograms of melatonin, serotonin, histamine and dopamine are shown below: Figure 2 The results are shown in Table 8 below; the chromatograms of melatonin, serotonin, histamine and dopamine at the lowest point of the standard curve are shown in the table. Figure 3-6 The standard curves of melatonin, serotonin, histamine and dopamine are shown in Figure 7-10 As shown, y is the ratio of the peak area of ​​the analyte to the corresponding internal standard peak area, and x is the concentration of the analyte.

[0144] Table 8

[0145]

[0146] Example 2

[0147] Blood samples were collected from 29 subjects and the levels of melatonin, serotonin, histamine, and dopamine were measured according to the following steps.

[0148] Step 1. Accurately aspirate 100 μL of plasma (or serum) sample and add 10 μL of internal standard solution (isotope internal standards of melatonin, serotonin, histamine, and dopamine: melatonin-D4, 5-hydroxytryptamine-D4, histamine-D4, and dopamine-D4, with concentrations of 0.05 ng / mL, 50 ng / mL, 1.5 ng / mL, and 0.5 ng / mL, respectively).

[0149] Step 2: Add 200 μL of zinc sulfate aqueous solution with a concentration of 200 mM, vortex to mix, centrifuge at high speed, and remove the supernatant.

[0150] Step 3: Add 100 μl of 2000 mM sodium carbonate aqueous solution to the supernatant in step 2 and vortex to mix.

[0151] Step 4: Add 100 μl of acetonitrile solution of benzoyl chloride (concentration is 10%), vortex mix, and centrifuge at high speed.

[0152] Step 5: Add 100 μl of 2 M formic acid aqueous solution to the supernatant in step 4, vortex to mix, and centrifuge at high speed.

[0153] Step 6: Load the supernatant from step 5 onto a C18-based SPE plate, wash, and then add 200 μL of 100% acetonitrile for elution.

[0154] Step 7: Inject into LC-MS for analysis.

[0155] The liquid phase conditions are as follows:

[0156] Chromatographic conditions:

[0157] Mobile phase A: methanol

[0158] Mobile phase B: 0.1% formic acid in water

[0159] The gradient conditions are the same as those in Table 6 of Example 1.

[0160] The mass spectrometry conditions are the same as those in Table 7 of Example 1.

[0161] The test results are shown in Table 9:

[0162] Table 9

[0163]

[0164]

[0165] It can be confirmed from Table 9 that the measurement results are highly consistent with the internationally recognized contents of melatonin, histamine, dopamine, and serotonin, proving that this method can accurately measure melatonin, histamine, dopamine, and serotonin without being interfered by interfering substances.

[0166]

[0167] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A neurotransmitter detection method based on an integrated derivatization and solid phase extraction continuous reaction platform, characterized in that: The neurotransmitters include at least two categories: Class A neurotransmitters include melatonin; Class B neurotransmitters include serotonin, histamine, and dopamine; Before the test, the neurotransmitters in the plasma or serum samples were derivatized using an aqueous derivatization system containing benzoyl chloride, and a 1M-3M formic acid aqueous solution was added to the obtained supernatant, mixed and centrifuged, and the obtained supernatant was subjected to LC-MS analysis after solid phase extraction; the volume ratio of the sample to the formic acid aqueous solution was 100:30-100; The derivatization treatment comprises the following steps: A1. Take a plasma or serum sample, add an internal standard solution and a 100-300 mM zinc sulfate aqueous solution, mix well, centrifuge and take the supernatant; the volume ratio of the sample to the zinc sulfate aqueous solution is 100:100-400; A2, add sodium carbonate aqueous solution with a concentration of 1000mM-2000mM and mix well; the volume ratio of sample to sodium carbonate aqueous solution is 100:50-100; A3. Continue to add 10%-20% benzoyl chloride acetonitrile solution, mix and centrifuge; the volume ratio of the sample to the benzoyl chloride acetonitrile solution is 100:20-100; The solid phase extraction is to load the obtained supernatant onto a C18-based SPE plate, and after elution, add 100% acetonitrile for elution; When performing LC-MS analysis, the liquid phase conditions are as follows: mobile phase A: methanol, mobile phase B: 0.1% formic acid aqueous solution, gradient conditions: 。 2. The neurotransmitter detection method according to claim 1, characterized in that: The internal standard in the internal standard solution is an isotope internal standard of a neurotransmitter.

3. The neurotransmitter detection method according to claim 1, characterized in that: Combined with LC-MS detection, the content of neurotransmitters in the samples was calculated based on the standard curve method.

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